J. Pineal Res. 2014; 56:398–407
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Molecular, Biological, Physiological and Clinical Aspects of Melatonin
Doi:10.1111/jpi.12127
Journal of Pineal Research
Melatonin inhibits MUC5AC production via suppression of MAPK signaling in human airway epithelial cells Abstract: Mucus acts as a primary defense system in the airway against various stimuli. However, excess mucus production causes a reduction in lung function via limitation of the airflow in the airway of patients suffering from asthma or chronic obstructive pulmonary disease (COPD). In this study, we evaluated the effects of melatonin on the production of MUC5AC, a major constituent of the mucin that is secreted from the airway, using epidermal growth factor (EGF)-stimulated NCI-H292 cells, a human mucoepidermoid carcinoma cell line, and an ovalbumin (OVA)-induced asthma murine model. Melatonin treatment significantly reduced the mRNA and protein levels of MUC5AC and reduced interleukin (IL)-6 production in EGF-stimulated H292 cells. Melatonin markedly decreased the phosphorylation of MAPKs, including ERK1/2, JNK, and p-38, induced by EGF stimulation. These findings were consistent with the results using MAPK inhibitors. Particularly, co-treatment with melatonin and a MAPK inhibitor more effectively suppressed MAPK phosphorylation than treatment with a MAPK inhibitor alone, which resulted in a reduction in MUC5AC expression. In the asthma murine model, melatonin-treated mice exhibited a marked reduction in MUC5AC expression in the airway compared with the OVA-induced mice. These reductions were accompanied by reductions in proinflammatory cytokine production and inflammatory cell infiltration. Collectively, these findings indicate that melatonin effectively inhibits MUC5AC expression. These effects may be closely associated with the inhibition of MAPK phosphorylation. Furthermore, our study suggests that melatonin could represent a potential therapeutic for chronic airway diseases, such as asthma and COPD.
Introduction The incidence of asthma and chronic obstructive pulmonary disease (COPD) and other economically important pulmonary diseases has increased in recent decades [1,2]. Mucus hypersecretion is principal characteristic in the pathogenesis of asthma and COPD and is closely associated with a reduction in lung function [3,4]. Mucins are major components of the mucus that are synthesized by epithelial goblet cells and the submucosal glands. Mucin 5AC (MUC5AC) is one of the primary mucin genes and is expressed predominantly in respiratory epithelium, constituting 95–98% of the mucin secreted in the airway [5]. In the host defense system, mucin plays a protective role for airway epithelial cells against various stimuli. However, because of its high viscosity, mucin is likely to cause airway obstruction if it is secreted in excess [6]. MUC5AC expression is induced by a variety of stimuli, including proinflammatory cytokines such as interleukin (IL)-1b and tumor necrosis factor (TNF)-a, proteins such as elastase and epidermal growth factor (EGF), and toxic bacterial products. In particular, the EGF receptor (EGFR) is activated by these stimuli and phosphorylated MAPK including 398
In-Sik Shin1,2, Ji-Won Park1,3, Na-Rae Shin1, Chan-Mi Jeon1, Ok-Kyoung Kwon1, Mee-Young Lee4, Hui-Seong Kim1, JongChoon Kim2, Sei-Ryang Oh1 and Kyung-Seop Ahn1 1
Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Chungbuk, Korea; 2College of Veterinary Medicine, Chonnam National University, Gwangju, Korea; 3College of Life Sciences and Biotechnology, Korea University, Seoul, Korea; 4Basic Herbal Medicine Research Group, Korea Institute of Oriental Medicine, Dajeon, Korea
Key words: asthma, MAPKs, melatonin, MUC5AC, NCI-H292 Address reprint requests to Kyung Seop Ahn, Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, 30 Yeongudanji-ro, Ochang-eup, Cheongwon-gun, Chungbuk 363-883, Korea. E-mail: [email protected] Received January 12, 2014; Accepted February 14, 2014.
c-Jun-N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p-38 [7]. These events lead to a rise in MUC5AC production. Indeed, various therapeutics that inhibit mucin secretion in the airway have been developed based on their ability to suppress MAPK phosphorylation [8,9]. Melatonin is an indoleamine produced by the pineal gland that exerts various biological effects, such as antioxidant, anticancer, anti-inflammatory, immunomodulatory, and neurobiological functions [10–15]. These findings have demonstrated the ability of melatonin to prevent damage by a variety of stimuli. Recent studies have demonstrated that melatonin inhibited the phosphorylation of MAPKs, including JNK, ERK, and p-38 in various experiments [16–18]. Nonetheless, it is uncertain whether melatonin induces its inhibitory effect on MUC5AC production via the suppression of phosphorylation of MAPKs. To address these questions, we examined the changes in MUC5AC production due to melatonin treatment in EGF-stimulated NCI-H292 cells, a line of human airway epithelial cells. Additionally, we treated the cells with MAPK inhibitors, including PD098059 (an ERK inhibitor), SP600125 (a JNK inhibitor), and SP600125 (a p-38
Effects of melatonin on MUC5AC production inhibitor) to clarify the role of melatonin in the phosphorylation of MAPKs in the modulation of MUC5AC production. Furthermore, we evaluated the effects of melatonin on MUC5AC production using a murine model of ovalbumin (OVA)-induced asthma.
Materials and methods Cell culture NCI-H292 cells, a human airway epithelial cell line, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) in the presence of penicillin (100 U/mL), streptomycin (100 lg/mL), and HEPES (25 mM) and incubated in a 5% CO2 incubator at 37°C. The cells were seeded on 96well plates at a density of 5 9 104 cells/well and incubated in serum-free medium in the presence various different concentrations of melatonin. After incubation for 24 hr, the cell viability was evaluated via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. All of the experiments were performed in triplicate. The effects of melatonin on MUC5AC expression The cells were seeded on 6-well plates at a density of 5 9 105 cells/well, treated with a nontoxic concentration of melatonin, and incubated in the presence of EGF (24 ng/mL). To investigate the effect of melatonin on MUC5AC expression, the cells were treated with melatonin (50, 100, 200, and 400 lM). The cells were harvested 24 hr after melatonin treatment. The total RNA was isolated using TrizolTM reagent (Invitrogen, Carlsbad, CA, USA) as instructed by the manufacturer, and a reverse transcription reaction was performed using a cDNA kit (Qiagen, Hilden, Germany) to investigate the effect of melatonin on expression of MUC5AC mRNA. Polymerase chain reactions were performed using specific forward and reverse primers (MUC5AC, forward, 50 -TGATCATCCAGCAGCAGGGCT, reverse, 50 -CCGAGCTCAGAGGACATATGGG; b-actin, forward, 50 -CATGTACGTTG CTATCCAGGC, reverse, 50 -CTCCTTAATGTCACGCACGAT) and a premixed solution according to the manufacturer’s instructions (Bioneer, Daejeon, Korea). MUC5AC protein was measured using an enzyme-linked immunosorbent assay (ELISA) kit (MyBioSource, San Diego, CA, USA). The absorbance at 450 nm was measured using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The effect of melatonin on IL-6 production The concentrations of IL-6 in the cultured supernatants were quantified using a commercial sandwich ELISA kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions. The absorbance at 450 nm was measured using a microplate reader (Bio-Rad Laboratories). The absolute concentrations were determined via comparison to standard curves generated using the same ELISA plates.
The effect of melatonin on the expression of MAPKs The cells were treated with various concentrations of melatonin, followed by incubation in the presence of EGF (25 ng/mL) for 30 min. The cells were collected via centrifugation, washed twice with phosphate-buffered saline (PBS), and resuspended in an extraction lysis buffer (Sigma-Aldrich, St Louis, MO, USA) containing protease inhibitors. The protein concentration was determined using a protein assay reagent (Bio-Rad Laboratories) according to the manufacturer’s instructions. Equal amounts of total cellular protein (30 lg) were resolved via electrophoresis using 12% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinyl difluoride membrane. The membrane was incubated in blocking solution (5% skim milk), followed by incubation overnight at 4°C in the appropriate primary antibody. The following primary antibodies and dilutions were used: ERK (1:2000 dilution; Cell Signaling, Danvers, MA, USA), pERK (1:1000 dilution; Cell Signaling), JNK (1:1000 dilution; Santa Cruz, Santa Cruz, CA, USA), pJNK (1:1000 dilution; Santa Cruz), p38 (1:1000 dilution; Santa Cruz), pp38 (1:1000 dilution; Santa Cruz), and b-actin (1:1000 dilution; Cell Signaling). The blots were washed three times with Tris-buffered saline containing Tween-20 (TBST) and then incubated in a 1:3000 dilution of a horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson Immunoresearch, West Grove, PA, USA) for 30 min at room temperature. The blots were again washed three times with TBST and then developed using an enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific, San Jose, CA, USA). The effects of melatonin on MAPK signaling To investigate the effects of melatonin on MAPK signaling in EGF-stimulated cells, the cells were pretreated with melatonin (400 lM) and MAPK inhibitors, including PD098059 (ERK inhibitor, 10 lM, Millipore Co., Billerica, MA, USA), SP600125 (JNK inhibitor, 10 lM, Millipore Co.), and SP600125 (p-38 inhibitor, 10 lM, Millipore Co.) and incubated for 30 min or 24 hr in presence of EGF (25 ng). The proteins were extracted as described above, and the supernatants were used in ELISA. The MAPK signaling was investigated via Western blot. The MUC5AC mRNA and protein levels were evaluated via RT-PCR and ELISA, respectively. Ovalbumin-induced asthma model Specific pathogen-free female BALB/c mice (6 wk old) were purchased from the Koatech Co. (Pyeongtaek, Korea) and used after 2 wk of quarantine and acclimatization. All of the experimental procedures were approved by the Institutional Animal Care and Use Committee of the Korea Research Institute of Bioscience and Biotechnology. The mice were sensitized on days 0 and 14 with an intraperitoneal injection of 20 lg of OVA (Sigma-Aldrich) emulsified with 2 mg of aluminum hydroxide in 200 lL of PBS (pH 7.4). On days 21, 22, and 23, the mice were subjected to airway challenge using OVA (1% (w/v)) for 1 hr 399
Shin et al. using an ultrasonic nebulizer (NE-U12; Omron Corp., Tokyo, Japan). Melatonin was administered to the mice via intraperitoneal injection at dose of 15 mg/kg body weight 1 hr prior to the OVA challenge. Dexamethasone, used as a positive control, was administered to the mice at a dose of 3 mg/kg body weight. The airway responsiveness was indirectly assessed 24 hr after the final airway challenge via single-chamber, whole-body plethysmography (Allmedicus, Seoul, Korea). Bronchoalveolar lavage fluid (BALF) samples were collected for analysis. The mice were sacrificed 48 hr after the final challenge via an intraperitoneal injection of pentobarbital (50 mg/kg; Hanlim Pharm. Co., Seoul, Korea), and a tracheostomy was performed. To obtain the BALF, ice-cold PBS (0.5 mL) was infused into the lungs three times and withdrawn each time using a tracheal cannula (a total volume 1.5 mL). The total number of inflammatory cells was determined by counting the cells in at least five squares of a hemocytometer after exclusion of the dead cells using Trypan blue staining. The differential cell counts in the BALF were performed using the Diff-Quikâ staining reagent (B41321A; IMEB Inc., San Marcos, CA, USA), according to the manufacturer’s instructions.
(A)
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Measurement of cytokines in the BALF and IgE in serum The levels of IL-4, IL-5, and IL-13 in the BALF were measured using ELISA kits (R&D System, Minneapolis, MN, USA) according to the manufacturer’s protocols. The levels of total IgE and OVA-specific IgE in serum were measured via ELISA (BioLegend, San Diego, CA, USA). For the measurement of OVA-specific IgE, 96-well microtiter plates were coated overnight with 10 lg/mL OVA in PBSTween-20. After washing and blocking each plate, the samples were incubated for 2 hr. Subsequently, the 96-well plates were washed, and a HRP-conjugated goat antimouse IgE antibody was added. After washing four times, 200 lL of o-phenylenediamine dihydrochloride (SigmaAldrich) was added to each well. Each plate was incubated for 10 min in the dark, and then the absorbance at 450 nm was determined using a microplate ELISA reader (Bio-Rad Laboratories). Measurement of the expression of MAPK signaling in lung tissue from OVA-challenged mice Lung tissue was homogenized (1/10 w/v) using a homogenizer with a tissue lysis/extraction reagent (Sigma-Aldrich) containing a protease inhibitor cocktail (Sigma-Aldrich). Each protein concentration was determined using Bradford reagent (Bio-Rad). Western blotting was performed as described above, and the phosphorylation of MAPK signaling including ERK, JNK, and p-38 was determined. Histology and immunohistochemistry After the BALF samples were collected, the lung tissue was fixed using 4% (v/v) paraformaldehyde. The tissues were paraffin-embedded, sectioned at a thickness of 4 lm, and stained using an H&E solution (Sigma-Aldrich) or the 400
Fig. 1. Melatonin reduces MUC5AC expression and IL-6 production in EGF-stimulated H292 cells. The expression of MUC5AC mRNA (A) and protein (B) was determined via RT-PCR and ELISA, respectively. Additionally, the production of IL-6 (C) was measured via ELISA. H292 cells were treated with melatonin for 1 hr, followed by epidermal growth factor (EGF) (25 ng/mL) for 24 hr. The expression of MUC5AC and the production of IL-6 were increased in EGF-stimulated H292 cells but were significantly decreased in melatonin-treated H292 cells in a concentration-dependent manner compared with the EGF-stimulated H292 cells. The data are presented as the means S.D. **P < 0.01 compared with the EGF-stimulated H292 cells.
periodic acidSchiff (PAS) solution (IMEB Inc.) to estimate the amount of inflammation or mucus production, respectively. For IHC, the paraffin-embedded sections were deparaffinized, dehydrated, washed using PBS with 0.3% Triton X-100, and preincubated for 10 min at room temperature with 10% goat serum to block nonspecific staining. Subsequently, slides were overnight at 4°C with primary mouse–rabbit MUC5AC antibody (1:100 dilution; Santa Cruz). After removing the primary antibodies, the sections were washed and incubated with biotinylated secondary antibody at 37°C for 1 hr, followed by incubation with avidin-biotin-peroxidase complex (Vector Laboratories,
Effects of melatonin on MUC5AC production (A)
(B)
(C)
Fig. 2. Melatonin decreased the phosphorylation of MAPKs including Jun-N-terminal kinase (JNK) (A), extracellular signal-regulated kinase (ERK) (B), and p-38 (C), in the EGF-stimulated H292 cells. H292 cells were treated with melatonin for 1 hr, followed by epidermal growth factor (EGF) (25 ng/mL) for 30 min. The expression of MAPKs expression was detected via Western blot. Quantification of p-JNK, p-ERK, and p-p38 expression was measured by Chemi-Doc (Bio-Rad, CA, USA). The EGF-stimulated H292 cells exhibited a significant elevation in p-JNK, p-ERK, and p-38 protein compared with the nonstimulated H292 cells. However, the melatonin-treated cells displayed a significant reduction in the phosphorylation of each MAPK in a concentration-dependent manner compared with the EGF-stimulated H292 cells. The JNK, ERK, and p38 expressions levels were used as loading controls for p-JNK, p-ERK, and p-p38, respectively. The data are presented as the means S.D. **P < 0.01 compared with the EGF-stimulated H292 cells.
Burlingame, CA, USA) for 1 hr at room temperature. The excess complex was removed and sections were washed with PBS prior to incubation in 0.05% diaminobenzidine (1:200; Millipore Co.) for a further 10 min. The sections were counterstained, rinsed with PBS to terminate the reaction, and protected with coverslips prior to microscopic examination. Statistical analysis The data are expressed as the means standard error of the mean (S.E. M.). Statistical significance was determined using analysis of variance (ANOVA) followed by a multiple comparison test using Dunnett’s test. P-values
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Molecular, Biological, Physiological and Clinical Aspects of Melatonin
Doi:10.1111/jpi.12127
Journal of Pineal Research
Melatonin inhibits MUC5AC production via suppression of MAPK signaling in human airway epithelial cells Abstract: Mucus acts as a primary defense system in the airway against various stimuli. However, excess mucus production causes a reduction in lung function via limitation of the airflow in the airway of patients suffering from asthma or chronic obstructive pulmonary disease (COPD). In this study, we evaluated the effects of melatonin on the production of MUC5AC, a major constituent of the mucin that is secreted from the airway, using epidermal growth factor (EGF)-stimulated NCI-H292 cells, a human mucoepidermoid carcinoma cell line, and an ovalbumin (OVA)-induced asthma murine model. Melatonin treatment significantly reduced the mRNA and protein levels of MUC5AC and reduced interleukin (IL)-6 production in EGF-stimulated H292 cells. Melatonin markedly decreased the phosphorylation of MAPKs, including ERK1/2, JNK, and p-38, induced by EGF stimulation. These findings were consistent with the results using MAPK inhibitors. Particularly, co-treatment with melatonin and a MAPK inhibitor more effectively suppressed MAPK phosphorylation than treatment with a MAPK inhibitor alone, which resulted in a reduction in MUC5AC expression. In the asthma murine model, melatonin-treated mice exhibited a marked reduction in MUC5AC expression in the airway compared with the OVA-induced mice. These reductions were accompanied by reductions in proinflammatory cytokine production and inflammatory cell infiltration. Collectively, these findings indicate that melatonin effectively inhibits MUC5AC expression. These effects may be closely associated with the inhibition of MAPK phosphorylation. Furthermore, our study suggests that melatonin could represent a potential therapeutic for chronic airway diseases, such as asthma and COPD.
Introduction The incidence of asthma and chronic obstructive pulmonary disease (COPD) and other economically important pulmonary diseases has increased in recent decades [1,2]. Mucus hypersecretion is principal characteristic in the pathogenesis of asthma and COPD and is closely associated with a reduction in lung function [3,4]. Mucins are major components of the mucus that are synthesized by epithelial goblet cells and the submucosal glands. Mucin 5AC (MUC5AC) is one of the primary mucin genes and is expressed predominantly in respiratory epithelium, constituting 95–98% of the mucin secreted in the airway [5]. In the host defense system, mucin plays a protective role for airway epithelial cells against various stimuli. However, because of its high viscosity, mucin is likely to cause airway obstruction if it is secreted in excess [6]. MUC5AC expression is induced by a variety of stimuli, including proinflammatory cytokines such as interleukin (IL)-1b and tumor necrosis factor (TNF)-a, proteins such as elastase and epidermal growth factor (EGF), and toxic bacterial products. In particular, the EGF receptor (EGFR) is activated by these stimuli and phosphorylated MAPK including 398
In-Sik Shin1,2, Ji-Won Park1,3, Na-Rae Shin1, Chan-Mi Jeon1, Ok-Kyoung Kwon1, Mee-Young Lee4, Hui-Seong Kim1, JongChoon Kim2, Sei-Ryang Oh1 and Kyung-Seop Ahn1 1
Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Chungbuk, Korea; 2College of Veterinary Medicine, Chonnam National University, Gwangju, Korea; 3College of Life Sciences and Biotechnology, Korea University, Seoul, Korea; 4Basic Herbal Medicine Research Group, Korea Institute of Oriental Medicine, Dajeon, Korea
Key words: asthma, MAPKs, melatonin, MUC5AC, NCI-H292 Address reprint requests to Kyung Seop Ahn, Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, 30 Yeongudanji-ro, Ochang-eup, Cheongwon-gun, Chungbuk 363-883, Korea. E-mail: [email protected] Received January 12, 2014; Accepted February 14, 2014.
c-Jun-N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p-38 [7]. These events lead to a rise in MUC5AC production. Indeed, various therapeutics that inhibit mucin secretion in the airway have been developed based on their ability to suppress MAPK phosphorylation [8,9]. Melatonin is an indoleamine produced by the pineal gland that exerts various biological effects, such as antioxidant, anticancer, anti-inflammatory, immunomodulatory, and neurobiological functions [10–15]. These findings have demonstrated the ability of melatonin to prevent damage by a variety of stimuli. Recent studies have demonstrated that melatonin inhibited the phosphorylation of MAPKs, including JNK, ERK, and p-38 in various experiments [16–18]. Nonetheless, it is uncertain whether melatonin induces its inhibitory effect on MUC5AC production via the suppression of phosphorylation of MAPKs. To address these questions, we examined the changes in MUC5AC production due to melatonin treatment in EGF-stimulated NCI-H292 cells, a line of human airway epithelial cells. Additionally, we treated the cells with MAPK inhibitors, including PD098059 (an ERK inhibitor), SP600125 (a JNK inhibitor), and SP600125 (a p-38
Effects of melatonin on MUC5AC production inhibitor) to clarify the role of melatonin in the phosphorylation of MAPKs in the modulation of MUC5AC production. Furthermore, we evaluated the effects of melatonin on MUC5AC production using a murine model of ovalbumin (OVA)-induced asthma.
Materials and methods Cell culture NCI-H292 cells, a human airway epithelial cell line, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) in the presence of penicillin (100 U/mL), streptomycin (100 lg/mL), and HEPES (25 mM) and incubated in a 5% CO2 incubator at 37°C. The cells were seeded on 96well plates at a density of 5 9 104 cells/well and incubated in serum-free medium in the presence various different concentrations of melatonin. After incubation for 24 hr, the cell viability was evaluated via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. All of the experiments were performed in triplicate. The effects of melatonin on MUC5AC expression The cells were seeded on 6-well plates at a density of 5 9 105 cells/well, treated with a nontoxic concentration of melatonin, and incubated in the presence of EGF (24 ng/mL). To investigate the effect of melatonin on MUC5AC expression, the cells were treated with melatonin (50, 100, 200, and 400 lM). The cells were harvested 24 hr after melatonin treatment. The total RNA was isolated using TrizolTM reagent (Invitrogen, Carlsbad, CA, USA) as instructed by the manufacturer, and a reverse transcription reaction was performed using a cDNA kit (Qiagen, Hilden, Germany) to investigate the effect of melatonin on expression of MUC5AC mRNA. Polymerase chain reactions were performed using specific forward and reverse primers (MUC5AC, forward, 50 -TGATCATCCAGCAGCAGGGCT, reverse, 50 -CCGAGCTCAGAGGACATATGGG; b-actin, forward, 50 -CATGTACGTTG CTATCCAGGC, reverse, 50 -CTCCTTAATGTCACGCACGAT) and a premixed solution according to the manufacturer’s instructions (Bioneer, Daejeon, Korea). MUC5AC protein was measured using an enzyme-linked immunosorbent assay (ELISA) kit (MyBioSource, San Diego, CA, USA). The absorbance at 450 nm was measured using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The effect of melatonin on IL-6 production The concentrations of IL-6 in the cultured supernatants were quantified using a commercial sandwich ELISA kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions. The absorbance at 450 nm was measured using a microplate reader (Bio-Rad Laboratories). The absolute concentrations were determined via comparison to standard curves generated using the same ELISA plates.
The effect of melatonin on the expression of MAPKs The cells were treated with various concentrations of melatonin, followed by incubation in the presence of EGF (25 ng/mL) for 30 min. The cells were collected via centrifugation, washed twice with phosphate-buffered saline (PBS), and resuspended in an extraction lysis buffer (Sigma-Aldrich, St Louis, MO, USA) containing protease inhibitors. The protein concentration was determined using a protein assay reagent (Bio-Rad Laboratories) according to the manufacturer’s instructions. Equal amounts of total cellular protein (30 lg) were resolved via electrophoresis using 12% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinyl difluoride membrane. The membrane was incubated in blocking solution (5% skim milk), followed by incubation overnight at 4°C in the appropriate primary antibody. The following primary antibodies and dilutions were used: ERK (1:2000 dilution; Cell Signaling, Danvers, MA, USA), pERK (1:1000 dilution; Cell Signaling), JNK (1:1000 dilution; Santa Cruz, Santa Cruz, CA, USA), pJNK (1:1000 dilution; Santa Cruz), p38 (1:1000 dilution; Santa Cruz), pp38 (1:1000 dilution; Santa Cruz), and b-actin (1:1000 dilution; Cell Signaling). The blots were washed three times with Tris-buffered saline containing Tween-20 (TBST) and then incubated in a 1:3000 dilution of a horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson Immunoresearch, West Grove, PA, USA) for 30 min at room temperature. The blots were again washed three times with TBST and then developed using an enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific, San Jose, CA, USA). The effects of melatonin on MAPK signaling To investigate the effects of melatonin on MAPK signaling in EGF-stimulated cells, the cells were pretreated with melatonin (400 lM) and MAPK inhibitors, including PD098059 (ERK inhibitor, 10 lM, Millipore Co., Billerica, MA, USA), SP600125 (JNK inhibitor, 10 lM, Millipore Co.), and SP600125 (p-38 inhibitor, 10 lM, Millipore Co.) and incubated for 30 min or 24 hr in presence of EGF (25 ng). The proteins were extracted as described above, and the supernatants were used in ELISA. The MAPK signaling was investigated via Western blot. The MUC5AC mRNA and protein levels were evaluated via RT-PCR and ELISA, respectively. Ovalbumin-induced asthma model Specific pathogen-free female BALB/c mice (6 wk old) were purchased from the Koatech Co. (Pyeongtaek, Korea) and used after 2 wk of quarantine and acclimatization. All of the experimental procedures were approved by the Institutional Animal Care and Use Committee of the Korea Research Institute of Bioscience and Biotechnology. The mice were sensitized on days 0 and 14 with an intraperitoneal injection of 20 lg of OVA (Sigma-Aldrich) emulsified with 2 mg of aluminum hydroxide in 200 lL of PBS (pH 7.4). On days 21, 22, and 23, the mice were subjected to airway challenge using OVA (1% (w/v)) for 1 hr 399
Shin et al. using an ultrasonic nebulizer (NE-U12; Omron Corp., Tokyo, Japan). Melatonin was administered to the mice via intraperitoneal injection at dose of 15 mg/kg body weight 1 hr prior to the OVA challenge. Dexamethasone, used as a positive control, was administered to the mice at a dose of 3 mg/kg body weight. The airway responsiveness was indirectly assessed 24 hr after the final airway challenge via single-chamber, whole-body plethysmography (Allmedicus, Seoul, Korea). Bronchoalveolar lavage fluid (BALF) samples were collected for analysis. The mice were sacrificed 48 hr after the final challenge via an intraperitoneal injection of pentobarbital (50 mg/kg; Hanlim Pharm. Co., Seoul, Korea), and a tracheostomy was performed. To obtain the BALF, ice-cold PBS (0.5 mL) was infused into the lungs three times and withdrawn each time using a tracheal cannula (a total volume 1.5 mL). The total number of inflammatory cells was determined by counting the cells in at least five squares of a hemocytometer after exclusion of the dead cells using Trypan blue staining. The differential cell counts in the BALF were performed using the Diff-Quikâ staining reagent (B41321A; IMEB Inc., San Marcos, CA, USA), according to the manufacturer’s instructions.
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(B)
(C)
Measurement of cytokines in the BALF and IgE in serum The levels of IL-4, IL-5, and IL-13 in the BALF were measured using ELISA kits (R&D System, Minneapolis, MN, USA) according to the manufacturer’s protocols. The levels of total IgE and OVA-specific IgE in serum were measured via ELISA (BioLegend, San Diego, CA, USA). For the measurement of OVA-specific IgE, 96-well microtiter plates were coated overnight with 10 lg/mL OVA in PBSTween-20. After washing and blocking each plate, the samples were incubated for 2 hr. Subsequently, the 96-well plates were washed, and a HRP-conjugated goat antimouse IgE antibody was added. After washing four times, 200 lL of o-phenylenediamine dihydrochloride (SigmaAldrich) was added to each well. Each plate was incubated for 10 min in the dark, and then the absorbance at 450 nm was determined using a microplate ELISA reader (Bio-Rad Laboratories). Measurement of the expression of MAPK signaling in lung tissue from OVA-challenged mice Lung tissue was homogenized (1/10 w/v) using a homogenizer with a tissue lysis/extraction reagent (Sigma-Aldrich) containing a protease inhibitor cocktail (Sigma-Aldrich). Each protein concentration was determined using Bradford reagent (Bio-Rad). Western blotting was performed as described above, and the phosphorylation of MAPK signaling including ERK, JNK, and p-38 was determined. Histology and immunohistochemistry After the BALF samples were collected, the lung tissue was fixed using 4% (v/v) paraformaldehyde. The tissues were paraffin-embedded, sectioned at a thickness of 4 lm, and stained using an H&E solution (Sigma-Aldrich) or the 400
Fig. 1. Melatonin reduces MUC5AC expression and IL-6 production in EGF-stimulated H292 cells. The expression of MUC5AC mRNA (A) and protein (B) was determined via RT-PCR and ELISA, respectively. Additionally, the production of IL-6 (C) was measured via ELISA. H292 cells were treated with melatonin for 1 hr, followed by epidermal growth factor (EGF) (25 ng/mL) for 24 hr. The expression of MUC5AC and the production of IL-6 were increased in EGF-stimulated H292 cells but were significantly decreased in melatonin-treated H292 cells in a concentration-dependent manner compared with the EGF-stimulated H292 cells. The data are presented as the means S.D. **P < 0.01 compared with the EGF-stimulated H292 cells.
periodic acidSchiff (PAS) solution (IMEB Inc.) to estimate the amount of inflammation or mucus production, respectively. For IHC, the paraffin-embedded sections were deparaffinized, dehydrated, washed using PBS with 0.3% Triton X-100, and preincubated for 10 min at room temperature with 10% goat serum to block nonspecific staining. Subsequently, slides were overnight at 4°C with primary mouse–rabbit MUC5AC antibody (1:100 dilution; Santa Cruz). After removing the primary antibodies, the sections were washed and incubated with biotinylated secondary antibody at 37°C for 1 hr, followed by incubation with avidin-biotin-peroxidase complex (Vector Laboratories,
Effects of melatonin on MUC5AC production (A)
(B)
(C)
Fig. 2. Melatonin decreased the phosphorylation of MAPKs including Jun-N-terminal kinase (JNK) (A), extracellular signal-regulated kinase (ERK) (B), and p-38 (C), in the EGF-stimulated H292 cells. H292 cells were treated with melatonin for 1 hr, followed by epidermal growth factor (EGF) (25 ng/mL) for 30 min. The expression of MAPKs expression was detected via Western blot. Quantification of p-JNK, p-ERK, and p-p38 expression was measured by Chemi-Doc (Bio-Rad, CA, USA). The EGF-stimulated H292 cells exhibited a significant elevation in p-JNK, p-ERK, and p-38 protein compared with the nonstimulated H292 cells. However, the melatonin-treated cells displayed a significant reduction in the phosphorylation of each MAPK in a concentration-dependent manner compared with the EGF-stimulated H292 cells. The JNK, ERK, and p38 expressions levels were used as loading controls for p-JNK, p-ERK, and p-p38, respectively. The data are presented as the means S.D. **P < 0.01 compared with the EGF-stimulated H292 cells.
Burlingame, CA, USA) for 1 hr at room temperature. The excess complex was removed and sections were washed with PBS prior to incubation in 0.05% diaminobenzidine (1:200; Millipore Co.) for a further 10 min. The sections were counterstained, rinsed with PBS to terminate the reaction, and protected with coverslips prior to microscopic examination. Statistical analysis The data are expressed as the means standard error of the mean (S.E. M.). Statistical significance was determined using analysis of variance (ANOVA) followed by a multiple comparison test using Dunnett’s test. P-values
